Gas Permeable Material

ABSTRACT

The invention describes a gas permeable fluoropolymer and silicone material used in the construction of cell culture bags.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/937,434, filed Mar. 27, 2018, now U.S. patent Ser. No. 10/711,235,which is a continuation of U.S. patent application Ser. No. 14/976,372,filed Dec. 21, 2015, now U.S. Pat. No. 9,926,524, which claims benefitunder 35 U.S.C. § 119(e) to U.S. Ser. No. 62/095,116, entitled“FLUORINATED SILICONE FILM FOR OXYGEN PERMEABLE MATERIAL”, filed Dec.22, 2014, the contents of each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to a gas permeable material andapplication to a cell culture apparatus and a cell culture method.

BACKGROUND OF THE INVENTION

In vitro cell culture is the complex process by which cells are grownunder controlled conditions, generally outside of their naturalenvironment but as close to their natural in vivo conditions aspossible. In practice cell culture refers to the culturing of cellsderived from multi-cellular eukaryotes, especially animal cells.However, there are also cultures of plants, fungi, insects and microbes,including viruses, bacteria and protista.

In vitro cell culture provides material necessary for research andapplication in pharmacology, physiology, and toxicology. This includesbioprocessing and cell therapy where cell cultures are necessary.

Cells are grown and maintained at an appropriate temperature and gasmixture in a cell incubator. Typically, mammalian cells are incubated at37° C. with a pH maintained between 7.2 and 7.4. The pH is typicallycontrolled using a bicarbonate buffering system in the medium, inconjunction with an incubator atmosphere of approximately 5-7% carbondioxide by volume. The carbon dioxide reacts with the water to formcarbonic acid which in turn interacts with bicarbonate ions in themedium to form a buffering system which maintains the pH nearphysiological levels. Oxygen is essential for cellular metabolism andgrowth. Culture conditions can vary for each cell type, and variation ofconditions for a particular cell type can result in differentphenotypes.

A variety of cell types are grown in culture including connective tissuecells, skeletal, cardiac, epithelial cells, neural cells, endocrinecells, immune cells, lymphocytes, melanocytes, and many types of tumorcells. Similarly a variety of media are available depending on theparticular growth requirements of the cells and the growth conditions.

Commercially available gas permeable cell culture bags are currently astandard device format used for cell culture. Cell culture bags that areconstructed with gas permeable films advantageously provide a largesurface area for gas exchange while maintaining a closed system.Disposables also helps reduce the risk of contamination for the cellculture and for the environment.

Cell culture bags are commercially available from OriGen BiomedicalGroup (OriGen PermaLife™ Bags), Baxter (Lifecell® X-Fold™ related toU.S. Pat. Nos. 4,829,002, 4,937,194, 5,935,847, 6,297,046 B1), Medtronic(Si-Culture™, U.S. Pat. No. 5,686,304), Biovectra (VectraCell™), andAmerican Fluoroseal (VueLife™ Culture Bag System, covered by U.S. Pat.Nos. 4,847,462 and 4,945,203).

Gas permeable films should be selected based on a variety ofcharacteristics including gas permeability, moisture vapor transmission,capacity to be altered for desired cell interaction with cells, opticalclarity, physical strength, and the like. A wide variety of informationexists that describe the types of gas permeable materials that have beensuccessfully used for cell culture.

Among various kinds of rubbery materials, silicone rubbers arepreferable in most cases in respect of their electric properties, lowcost, precision moldability and durability in repeated bending andreleasing movements.

Silicone films for cell culture bags have high oxygen permeability, goodoptical clarity, good resistance to puncture, typically do not bindcells, and can be easily fabricated into a wide variety of shapes.Silicone films may be less than about 3 mm, about 2 mm, about 1 mm, orabout 0.8 mm in the surface areas where gas transfer is desired. Thebest selection of material depends on the application.

Fluoropolymer films have desirable characteristics that make them apopular choice for culture bags. Compared to silicone, fluoropolymerfilms are more biologically, chemically and immunologically inert, aswell as being hydrophobic. Fluoropolymer films like FEP (fluorinatedethylene-propylene) do not trigger immune responses in immune cells andprogenitor immune cells. However fluoropolymer films are more expensive,less gas permeable then silicone films, and are difficult to adhere toother polymers.

Therefore a need exists which alleviates the disadvantages of usingeither silicone or fluoropolymer for cell culture bags alone.

BRIEF SUMMARY OF THE INVENTION

In one aspect an oxygen permeable composition is provided. In anotheraspect a two-part composite having a first layer of elastomer and asecond fluid contact layer, wherein the second fluid contact layer has atotal organic carbon (TOC) in water of less than 1 mg/cm². The secondfluid contact layer having low TOC can be a fluoropolymer and the firstelastomer layer and the second fluoropolymer layer are adhered together.More particularly, the embodiments disclosed herein relate to afluoropolymer film capable of bonding to the surface of an elastomericrubber. The fluoropolymer coating exhibits a high bonding strength withor without the use of adhesives.

In another embodiment the outer elastomer layer is natural polyisopenerubber (NR), synthetic polyisoprene rubber (IR), polybutadiene rubber(BR), chloropene rubber (CR), butyl rubber (IIR), halogenated butylrubbers (CIIR, BIIR), styrene-butadiene rubber (SBR), nitrile rubber(NBR) and hydrogenated nitrile rubber (HNBR), ethylene propylene rubber(EPM), ethylene propylene diene rubber (EPDM), epichlorohydrin rubber(ECO), polyacrylic rubber (ACM, ABR), silicone rubber (SI, Q, VMQ),fluorosilicone rubber (FSR, FVMQ), fluoroelastomers (FKM, FEPM),perfluoroelastomers (FFKM), polyether block amides (PEBA),chlorosulfonated polyethylene (CSM), ethylene-vinyl acetate (EVA),cyclic olefin copolymers, polyolefin elastomers, elastomeric PET, ormixtures thereof.

In another embodiment the outer elastomer layer is polymethylpentenepolymer (PMP).

In another embodiment the oxygen permeable film comprises an outersilicone rubber layer and an inner fluoropolymer layer wherein the innerfluoropolymer layer is coated onto the outer silicone rubber layer.

In another embodiment the silicone rubber of the oxygen permeable filmis high consistency rubber (HCR), fluorosilicone rubber (FSR), liquidsilicone rubber (LSR), or room temperature vulcanized rubber (RTV),thermoplastic silicone rubber (TPE), platinum cured silicone rubber, orperoxide cured silicone rubber.

In another embodiment the silicone rubber of the oxygen permeable filmis thermoset, cured, vulcanized, catalyzed, or injection molded.

In another embodiment the fluoropolymer layer of the film is fluorinatedethylene-propylene (FEP), polytetrafluoroethylene (PTFE),tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidenedifluoride, hexafluoropropylene/tetrafluoroethylene/vinylidene copolymer(THV), or perfluoro(1-butenyl vinyl ether) homocyclopolymer havingfunctionalized polymer-end groups

In another embodiment the elastomer, such as PMP or silicone andfluoropolymer layers of the oxygen permeable film are bonded.

In still another embodiment the adhesion of the elastomer, such as PMPor silicone, and fluoropolymer layers of the oxygen permeable film is bycoating, RF welding, ultrasonic welding, hot bar welding, chemicalbonding, adhesive bonding, thermal fusion bonding, solvent welding,laser welding, corona discharge, radiation, lamination such as byextreme heat, belt, or melt lamination, etching, plasma treatment,wetting, adhesives, radiation, extrusion, co-extrusion, or combinationsthereof.

In another embodiment an oxygen permeable container comprises an outerelastomer layer and an inner fluid contact layer having low TOC. In oneaspect the fluid contact layer is a fluoropolymer layer, wherein theinner fluoropolymer layer is adhered to the outer elastomer layer of thecontainer.

In another embodiment the outer elastomer layer of the container isnatural polyisopene rubber (NR), synthetic polyisoprene rubber (IR),polybutadiene rubber (BR), chloropene rubber (CR), butyl rubber (IIR),halogenated butyl rubbers (CIIR, BIIR), styrene-butadiene rubber (SBR),nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR), ethylenepropylene rubber (EPM), ethylene propylene diene rubber (EPDM),epichlorohydrin rubber (ECO), polyacrylic rubber (ACM, ABR), siliconerubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomers(FKM, FEPM), perfluoroelastomers (FFKM), polyether block amides (PEBA),chlorosulfonated polyethylene (CSM), ethylene-vinyl acetate (EVA),cyclic olefin copolymers, polyolefin elastomers, elastomeric PET, orcombination thereof.

In another embodiment the outer elastomer layer of the container ispolymethylpentene polymer (PMP).

In another embodiment the outer elastomer layer of the container issilicone rubber.

In yet another embodiment the silicone rubber of the oxygen permeablecontainer is high consistency rubber (HCR), fluorosilicone rubber (FSR),liquid silicone rubber (LSR), or room temperature vulcanized rubber(RTV), thermoplastic silicone rubber (TPE), platinum cured siliconerubber, or peroxide cured silicone rubber.

In still another embodiment the silicone rubber of the oxygen permeablecontainer is thermoset, cured, vulcanized, catalyzed, or injectionmolded.

In another embodiment the fluoropolymer layer of the container isfluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE),polyvinylidenefluoride (PVDF), tetrafluoroethylene-perfluoro(propylvinyl ether) (PFA), polyvinylidene difluoride (PVF),polychlorotrifluoroethlylene (PCTFE),tetrafluoroethylene/hexafluoropropylene/ethylene copolymer (HTE),chlorotrifluoroethylene/vinylidenefluoride copolymer,chlorotrifluoroethylene/hexafluoropropylene,ethylene/chlorotrifluoroethylene copolymers (ECTFE),ethylene/trifluoroethylene copolymers, ethylene/tetrafluoroethylenecopolymers (ETFE), tetrafluoroethylene/propylene copolymers (TFE/P),tetrafluoroethylene/hexafluoropropylene copolymers (FEP/HFP),hexafluoropropylene/tetrafluoroethylene/vinylidene copolymer (THV), orperfluoro(1-butenyl vinyl ether) homocyclopolymer having functionalizedpolymer-end groups

In another embodiment the elastomer layer, such as a silicone or PMPlayer, and fluoropolymer layers of the oxygen permeable container arebonded.

In another embodiment the bonding of the oxygen permeable container isby coating, RF welding, ultrasonic welding, hot bar welding, chemicalbonding, adhesive bonding, thermal fusion bonding, solvent welding,laser welding, corona discharge, radiation, lamination such as byextreme heat, belt, or melt lamination, etching, plasma treatment,wetting, adhesives, radiation, extrusion, co-extrusion, or combinationsthereof.

In another embodiment the oxygen permeable container comprises a bag,flask, or tube.

In another embodiment the oxygen permeable bag, flask, or tube containsa sealable access port.

A method is provided to cultivate cells in an oxygen permeablecontainer. The method includes the steps of a) adding media to thecontainer and b) adding cells to the container wherein the containercomprises an outer elastomer layer, an inner fluid contact layer havinga TOC in water of less than 1 mg/cm². In one aspect the inner fluidcontact layer is a fluoropolymer layer, wherein the inner fluoropolymerlayer is coated onto the outer elastomer layer, and wherein the cellsand media are in contact with the inner fluoropolymer layer.

In another embodiment of the method, the outer elastomer layer of thecontainer is natural polyisopene rubber (NR), synthetic polyisoprenerubber (IR), polybutadiene rubber (BR), chloropene rubber (CR), butylrubber (IIR), halogenated butyl rubbers (CIIR, BIIR), styrene-butadienerubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber(HNBR), ethylene propylene rubber (EPM), ethylene propylene diene rubber(EPDM), epichlorohydrin rubber (ECO), polyacrylic rubber (ACM, ABR),silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ),fluoroelastomers (FKM, FEPM), perfluoroelastomers (FFKM), polyetherblock amides (PEBA), chlorosulfonated polyethylene (CSM), ethylene-vinylacetate (EVA), cyclic olefin copolymers, polyolefin elastomers,elastomeric PET, or combination thereof.

In another embodiment of the method, the outer elastomer layer of thecontainer is polymethylpentene polymer (PMP).

In another embodiment of the method, the outer elastomer layer of thecontainer is silicone rubber.

In yet another embodiment of the method, the silicone rubber of theoxygen permeable container is high consistency rubber (HCR),fluorosilicone rubber (FSR), liquid silicone rubber (LSR), or roomtemperature vulcanized rubber (RTV), thermoplastic silicone rubber(TPE), platinum cured silicone rubber, or peroxide cured siliconerubber.

In still another embodiment of the method, the silicone rubber of theoxygen permeable container is thermoset, cured, vulcanized, catalyzed,or injection molded.

In another embodiment of the method, the fluoropolymer layer of thecontainer is fluorinated ethylene-propylene (FEP),polytetrafluoroethylene (PTFE), 3M™ Dyneon™ TFM™ modified PTFE,polyvinylidenefluoride (PVDF), tetrafluoroethylene-perfluoro(propylvinyl ether) (PFA), polyvinylidene difluoride (PVF),polychlorotrifluoroethlylene (PCTFE),tetrafluoroethylene/hexafluoropropylene/ethylene copolymer (HTE),chlorotrifluoroethylene/vinylidenefluoride copolymer,chlorotrifluoroethylene/hexafluoropropylene,ethylene/chlorotrifluoroethylene copolymers (ECTFE),ethylene/trifluoroethylene copolymers, ethylene/tetrafluoroethylenecopolymers (ETFE), tetrafluoroethylene/propylene copolymers (TFE/P),tetrafluoroethylene/hexafluoropropylene copolymers (FEP/HFP),hexafluoropropylene/tetrafluoroethylene/vinylidene copolymer (THV), orperfluoro(1-butenyl vinyl ether) homocyclopolymer having functionalizedpolymer-end groups

In another embodiment of the method, the elastomer, e.g., PMP orsilicone, and fluoropolymer layers of the oxygen permeable container arebonded.

In another embodiment of the method, the bonding of the oxygen permeablecontainer is by chemical bonding, adhesive bonding, thermal fusionbonding, solvent bonding, laser welding, surface treatment, extrusion,co-extrusion, coating, lamination, belt lamination, coating, orcombinations thereof.

In another embodiment of the method, the oxygen permeable containercomprises a bag, flask, or tube.

In another embodiment of the method, the oxygen permeable bag, flask, ortube contains a sealable access port.

In another embodiment of the method to cultivate cells the steps a)adding media to the container and b) adding cells to the container areinterchangeable.

In another embodiment of the method to cultivate cells the steps a) andb) are followed by incubation of the container.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed descriptions are to be regarded asillustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a side view of a fluoropolymer coated elastomer film.

FIG. 2 shows a fluoropolymer coated elastomer culture container.

FIG. 3 shows a graphical representation of the initial ingress of oxygeninto incumbent FEP bag.

FIG. 4 shows a graphical representation of the initial ingress of oxygenin prototype bag with gas permeable laminate.

FIG. 5 shows a graphical representation of the approach to equilibriumin prototype bag made with gas permeable laminate.

DETAILED DESCRIPTION

In the specification and in the claims, the terms “including” and“comprising” are open-ended terms and should be interpreted to mean“including, but not limited to . . . . ” These terms encompass the morerestrictive terms “consisting essentially of” and “consisting of.”

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”,“characterized by” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications and patentsspecifically mentioned herein are incorporated by reference in theirentirety for all purposes including describing and disclosing thechemicals, instruments, statistical analyses and methodologies which arereported in the publications which might be used in connection with theinvention. All references cited in this specification are to be taken asindicative of the level of skill in the art. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

A combination of the beneficial properties of fluoropolymer andelastomer films described herein applied to cell culture bags is desiredin the field and provided herein.

The gas permeable film pertaining to a first aspect described hereincomprises a fluoropolymer coated elastomer membrane. Oxygen is necessaryfor cellular growth in a culture medium and carbon dioxide is necessaryfor the culture medium to buffer pH. Most normal mammalian cell linesgrow well at pH 7.4, and there is very little variability amongdifferent cell strains. However, some transformed cell lines have beenshown to grow better at slightly more acidic environments (pH 7.0-7.4),and some normal fibroblast cell lines prefer slightly more basicenvironments (pH 7.4-7.7). Because the pH of the medium is dependent onthe delicate balance of dissolved carbon dioxide and bicarbonate,changes in carbon dioxide concentration can alter the pH of the medium.Therefore it is necessary for a fluoropolymer coated elastomer membraneto have good gas permeability to oxygen and carbon dioxide for use in acell culture bag.

Those skilled in the art will recognize that the gas permeable materialshould be selected based on a variety of characteristics includingflexibility, sealability that ensures airtightness, good clarity thatpermits the microscopic examination of cell growth, freedom fromplasticizers (such as dioctyl phthalate and diisodecyl phthalate) thatcan be harmful to cells, moisture vapor transmission, capacity to bealtered for desired cell interaction with cells, optical clarity,physical strength, and the like.

Elastomers are polymers with viscoelasticity and very weakinter-molecular forces, generally having low Young's modulus and highfailure strain compared to other materials. The term elastomers may beused interchangeably with the term rubber, although rubber is preferredwhen referring to vulcanisates. Elastomers are amorphous polymersconstructed from monomers of carbon, hydrogen, oxygen, and/or silicon.

Elastomers comprise unsaturated rubbers that can be cured by sulfurvulcanization, for example natural (NR) and synthetic polyisoprene (IR),polybutadiene (BR), chloropene rubber (CR), butyl rubber (IIR),halogenated butyl rubbers (CIIR, BIIR), styrene-butadiene rubber (SBR),nitrile (NBR) and hydrogenated nitrile rubber (HNBR).

Elastomers comprise unsaturated rubbers that cannot be cured by sulfurvulcanization, for example ethylene propylene rubber (EPM), ethylenepropylene diene rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylicrubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber(FSR, FVMQ), fluoroelastomers (FKM, FEPM), perfluoroelastomers (FFKM),polyether block amides (PEBA), chlorosulfonated polyethylene (CSM),thermoplastic urethanes (TPUs), including thermoplastic silicones, suchas a GENIOMER®, cyclic olefin copolymers, polyolefin elastomers,elastomeric PET, and ethylene-vinyl acetate (EVA).

Thermoplastic polyurethanes (TPUs) are known in the art. Typically, athermoplastic polyurethane is formed by reacting a polyol with anisocyanate. The overall properties of the polyurethane will depend uponthe type of polyol and isocyanate, crystallinity in the polyurethane,the molecular weight of the polyurethane and chemical structure of thepolyurethane backbone.

Polyurethanes may be either thermoplastic or thermoset, depending on thedegree of crosslinking present. Thermoplastic urethanes (TPUs) do nothave primary crosslinking while thermoset polyurethanes have a varyingdegree of crosslinking, depending on the functionality of the reactants.

Thermoplastic polyurethanes are commonly based on either methylenediisocyanate (MDI) or toluene diisocyanate (TDI) and include bothpolyester and polyether grades of polyols. Thermoplastic polyurethanescan be formed by a “one-shot” reaction between isocyanate and polyol orby a “pre-polymer” system, wherein a curative is added to the partiallyreacted polyolisocyanate complex to complete the polyurethane reaction.Examples of some common thermoplastic polyurethane elastomers based on“pre-polymers” are “TEXIN”, a tradename of Bayer Materials Science,“ESTANE”, a tradename of Lubrizol, “PELLETHANE”, a tradename of DowChemical Co., and “ELASTOLLAN”, a tradename of BASF, Inc.

GENIOMER® thermoplastic silicones include, but are not limited toGENIOMER® 140 Silicone TPE, GENIOMER® 200 Silicone TPE Elastomer (90%polydimethylsiloxane and isocyanate), GENIOMER®, 60 Silicone TPE,GENIOMER® 80 Silicone TPE and GENIOMER® 145 TPE, all of which comprise90% polydimethylsiloxane and isocyanate.

Polymethylpentene (PMP) is a thermoplastic polymer of methylpentenemonomer units and is highly gas permeable and transparent.

Silicone rubber has proven to be a particularly good material for a gaspermeable membrane. To guarantee sufficient oxygen and carbon dioxideexchange, the thinnest possible gas exchange membranes are preferred.Membranes with a thickness between 0.1 mm and 1 mm have provensuccessful.

A silicone membrane may be manufactured economically in any desiredshape by injection molding. Silicone is available commercially in manythicknesses, shapes, and specific gas permeabilities. It has high tearresistance and good chemical resistance to the media ordinarily used incell culturing, and is therefore also especially easy to handle.

The ability to sterilize a gas permeable silicone membrane is alsoespecially advantageous. In particular, it can be effectively sterilizedin an autoclave with no substantial changes in shape and can be reusedseveral times.

It is preferred that the silicone rubber used has a leachable andextractable profile as low as possible.

In one embodiment a cell culture container may comprise a single layerof an elastomer, such as a thermoplastic silicone (e.g. GENIOMER® byWacker Chemie AG, a polydimethylsiloxane/urea copolymer) or afluoropolymer with TOC and permeability properties as described herein.

It should also be noted that other configurations of thermoplastics(elastomer and non-elastomer) and fluoropolymer configurations couldalso be used to control the gas permeability of a composite, whilstcontaining a low TOC fluid contact layer. Control of gas permeabilitycould be for purpose of either creating a high or low gas permeablecomposite. Examples of thermoplastics elastomers (TPE) include styreneblock copolymers (TPE-s), olefins (TPE-o), alloys (TPE-v or TPV),polyurethanes (TPU), copolyesters, and polyamides. Examples ofnon-elastomer thermoplastics include acrylics, acrylonitrile butadienestyrene (ABS), nylon, polylactic acid (PLA), polybenzimidazole (PBI),polycarbonate (PC), polyether sulfone (PES), polyetherether ketone(PEEK), polyetherimide (PEI), polyethylene (PE), polyphenylene oxide(PPO), polyphenylene sulfide (PPS), polypropylene (PP), polystyrene(PS), and polyvinyl chloride (PVC), ethylene vinyl alcohol (EVOH), aswell as any traditionally rigid polymer whose monomer architecture hasbeen modified to reduce crystallinity and increase flexibility.

Microporous, hydrophobic fluoropolymers, for example 3M™ Dyneon™ TFM™modified PTFE, HTE, or THV, have also proven advantageous as materialsfor the gas exchange membrane. The hydrophobic nature of fluoropolymersensures that the gas exchange membrane is impermeable to aqueous media.For a given gas permeability, the required geometry of the gas exchangemembrane depends on the gas requirement resulting for cell respiration,and on the partial pressures of the gases involved in cell respiration,especially on the oxygen partial pressure acting on it from outside.

FIG. 1. shows a side view of fluoropolymer coated elastomer film 1. Itshould be understood that fluoropolymer layer 10 can be disposed uponelastomer layer 20 to form film 1. Fluoropolymer layer 10 can come froma castable solution. Alternatively, the elastomer can also becast/disposed onto the fluoropolymer layer.

Typically, fluoropolymer layer 10 has a thickness from about 0.001 mm toabout 0.7 mm, more particularly from about 0.005 mm to about 0.4 mm andmost particularly from about 0.01 mm to about 0.1 mm and all thicknessesin between, including 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07mm, 0.08 mm, and 0.09 mm. In a particular aspect, the fluoropolymer is aFEP layer and the thickness of the fluoropolymer layer is 0.0254 mm or 1mil.

Elastomer layer 20 generally has a thickness of from about 0.01 mm toabout 5 mm, more particularly from about 0.05 mm to about 1 mm and mostparticularly from about 0.1 mm to about 0.5 mm and all thicknesses inbetween, including 0.15 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm,and 0.45 mm. In a particular aspect, the elastomer is a thermosetsilicone layer and the thickness of the elastomer layer is 0.127 mm to0.254 mm or 5 to 10 mil.

The term “castable” is intended to mean a fluoropolymer ornon-fluoropolymer capable of being dispersed, dissolved, suspended,emulsified or otherwise distributed in a liquid carrier medium. Theliquid carrier medium may be water, organic solvent, or any other liquidin which the polymer may be dispersed, dissolved, suspended, emulsifiedor otherwise distributed. The liquid carrier medium may be a mixture ofsuitable liquids. Once distributed within the carrier medium, thepolymer and medium is then capable of being deposited or cast upon asupporting material to form a film. The polymer(s) can be mixed with afirst carrier liquid. The mixture may comprise a dispersion of polymericparticles in the first carrier liquid, an emulsion of liquid droplets ofthe polymer, or of a monomeric or oligomeric precursor of the polymer inthe first carrier liquid or a solution of the polymer in the firstcarrier liquid. Additional/equivalent situations that would make apolymer “castable” would be if it is heated above its melting point orprocessed in a liquid state and then cured by UV, IR, initiatiors, orebeam so that it becomes solid.

The castable polymer(s) may also be a monomeric or oligomeric precursorof the polymer distributed within a carrier liquid. Most commonlycastable compositions are emulsions or dispersions in aqueous media.

The choice of the first carrier liquid is based on the particularpolymer and the form in which the material is to be introduced to thecasting composition of the present invention. If a solution is desired,a solvent for the particular fluoropolymer is chosen as the carrierliquid. Suitable carriers include, for example, DMAC, NMP, glycolethers, or water and the like. If a dispersion is desired, then asuitable carrier is one in which the polymer is not soluble. An aqueoussolution would be a suitable carrier liquid for a dispersion of polymerparticles.

Most commonly castable compositions are emulsions or dispersions inaqueous media. Surfactants can be used to prepare a dispersion in anamount effective to modify the surface tension of the carrier liquid.Suitable surfactant compounds include ionic surfactants, amphoteric,cationic and nonionic surfactants.

Fluoropolymers are generally selected as the fluid contact layer becauseof their hydrophobicity and because they are biologically, chemicallyand immunologically inert.

The phrase “fluoropolymer” is known in the art and is intended toinclude, for example, fluorinated ethylene-propylene (FEP),polytetrafluoroethylene (PTFE), polyvinylidenefluoride (PVDF),polychlorotrifluoroethlylene (PCTFE), polyvinylfluoride (PVF),tetrafluoroethylene/hexafluoropropylene/ethylene copolymer (HTE),chlorotrifluoroethylene/vinylidenefluoride copolymer,chlorotrifluoroethylene/hexafluoropropylene,ethylene/chlorotrifluoroethylene copolymers (ECTFE),ethylene/trifluoroethylene copolymers, ethylene/tetrafluoroethylenecopolymers (ETFE), tetrafluoroethylene/propylene copolymers (TFE/P),tetrafluoroethylene/hexafluoropropylene copolymers (FEP/HFP),tetrafluoroethylene-perfluoro(alkyl vinyl ether) copolymers (e.g., PFA;tetrafluoroethylene-perfluoro(propyl vinyl ether), polyvinylidenedifluoride, hexafluoropropylene/tetrafluoroethylene/vinylidenecopolymers (THV), and mixtures thereof.

The phrase “fluoropolymer” also includes amorphous polymers which arenon-crystalline when measured by DSC, or whose heat of melting is lessthan 2 J/g. These include copolymers of TFE with functional ornon-functional monomers such as fluoroolefins having 2-8 carbon atomsand fluorinated alkyl vinyl ether in which the alkyl group contains 1 or3 to 5 carbon atoms. Examples of the non-functional monomers includehexafluoropropylene (HFP), chlorotrifluoro ethylene (CTFE), PEVE, PMVEand perfluoro-(propylene vinyl ether) (PPVE). Functional monomersinclude perfluoroethyl vinyl ether (EVE), CF₂CFOCF₂CFCF₃OCF₂CF₂COOCH₃(EVE-carbamate), CF₂CFOCF₂CFCF₃OCF₂CF₂SO₂F (PSEPVE),CF₂CFOCF₂CFCF₃OCF₂CF₂CN (8CNVE), N₃(CF₂CFOCF₂CFCF₃OCF₂CF₂)₃(EVE-triazine), CF₂CFOCF₂CFCF₃OCF₂CF₂CN (EVE-CN), CF₂CFOCF₂CFCF₃OCF₂CF₂CH₂OH (EVE-OH), CF₂CFOCF₂CFCF₃OCF₂CF₂CH₂PO₂(OH)₂(EVE-P)CF₂CFOCF₂CFCF₃OCF₂CF₂CH₂COOH (EVE-COOH), and2,2-bistrifluoromethyl-4,5-difluoro-1,3-dioxole (PDD).

Commercially available amorphous fluoropolymer materials include thosefrom DuPont, Wilmington, Del.: TEFLON® SF60 (TFE/PMVE/PEVE, DuPont,Wilmington Del.), TEFLON® SF61 (TFE/PMVE/PEVE/EVE-P), TEFLON® SF50(TFE/HFP), Teflon® AF 1600 (PDD/TFE), and TEFLON® AF2130 (PDD/CTFE);from Asahi Corporation of Tokyo, Japan: CYTOP® (CYTOP type A, CYTOP typeM, CYTOP type S, or CYTOP NM); from MY Polymers Corporation of Rehovot,Israel (MY-133); or from Nusil Corporation of Carpinteria, Calif.(LS-233).

The fluoropolymer solutions may be applied by common coating methods,including but not limited to spray application, dip coating, brushing,or extrusion/injection molding.

CYTOP® is a perfluoro(1-butenyl vinyl ether) homocyclopolymer containingfunctionalized end groups. It exists as an amorphous resin and isavailable in a variety of concentrations in perfluorinated solvents andselect nonfluoro solvent systems. For example: 0.1 to 1% in 9:5isopropyl alcohol/isobutyl acetate.

CYTOP type A contains a carboxyl polymer-end group and CYTOP type Mcontains an amino-silane polymer-end group. Either fluoropolymer can beused with promoters, special primers, or a silane coupling agent forapplication as a plastic coating. CYTOP type S contains perfluoropolymer-end group for non-adhesion UV resistance and high transparency.

The silicone rubber formulation includes a silicone polymeric matrix.The polymeric matrix may be formed, for example, using a non-polarsilicone polymer. The silicone polymer may, for example, includepolyalkylsiloxanes, such as silicone polymers formed of a precursor,such as dimethylsiloxane, diethylsiloxane, dipropylsiloxane,methylethylsiloxane, methylpropylsiloxane, or combinations thereof. In aparticular, the polyalkylsiloxane includes a polydialkylsiloxane, suchas polydimethylsiloxane (PDMS). In general, the silicone polymer isnon-polar and is free of halide functional groups, such as chlorine andfluorine, and of phenyl functional groups.

Functionalized polyorganosiloxanes include polydimethylsiloxanes whichare endblocked by vinyldimethylsiloxy groups at both ends, dimethylsiloxane-vinylmethylsiloxane copolymers which are endblocked byvinyldimethylsiloxy groups at both ends, anddimethylsiloxane-methylphenyl siloxane copolymers which are endblockedby vinyldimethylsiloxy groups at both ends.

The silicone rubber may comprise a platinum catalyzed liquid siliconerubber (LSR) or a high consistency gum rubber (HCR). The silicone rubbermay also comprise a peroxide catalyzed silicone rubber (LSR) or a highconsistency gum rubber (HCR). For example, Saint-Gobain produces medicalproducts made from SILMEDIC®, a peroxide based silicone. An example ofthe silicone rubber as an HCR is GE 94506 HCR available from GEPlastics. Examples of LSR include Wacker 3003 by Wacker Silicone ofAdrian, Mich. and Rhodia 4360 by Rhodia Silicones of Ventura, Calif.

In one aspect of the invention, when the silicone rubber is employed asa base rubber, the composition of fluoropolymer coating may contain areactive compound which binds with the silicone rubber. Examples of thereactive compounds that bind to silicone rubber include hydrocarbonsilicon-containing compounds which are particularly preferred.

Examples of such silicon-containing compounds include compounds having asiloxane bond, silane coupling agents having an alkoxysilyl group,functional silanes having a chlorosilyl group or silazane, amino-silane,and silylating agents. Of these, compounds having a siloxane bond, andsilane coupling agents having an alkoxysilyl group are preferred.Examples of such compounds having a siloxane bond include end-modifieddimethylsiloxanes, condensation-type or addition-type liquid silicones,silicate salts, and acrylic silicone polymers.

An example of a fluoropolymer having an amino-silane end modification isCYTOP type M. Similarly, an example of a fluoropolymer having a carboxylgroup end modification is CYTOP type A. Either may be mixed with orwithout a coupling agent and bond directly or indirectly via couplingagent with a silicone rubber. Chemical bonding may occur bycondensation, alkylation, amidation, silylation, etherification, orthrough a coupling agent by any bonding combination thereof. Forexample, a coupling agent that may be used with silicone rubber is anisocyanate compound that reacts with hydroxyl, amino, or sulfur groups.After formation of a chemical bond between the fluoropolymer and thesilicone rubber, adhesion will be achieved and the composite gaspermeable membrane will have a higher strength with minimal leaching.Either CYTOP type A or CYTOP type M after hydrolysis may also adhere tosilicone rubber through hydrogen bonding.

Bonding of the elastomer layer and fluoropolymer layers together mayalso be accomplished by laser welding, surface treatment, radiation,extrusion, co-extrusion, coating, lamination, wetting, adhesives, orcombinations thereof. Preferred methods of surface treatment includeC-treatment (Saint-Gobain Performance Plastics Corporation, U.S. Pat.No. 6,726,979), corona discharge, plasma treatment, etching, orcombinations thereof. Surface treatments may also involve chemicaltreatment with additives or primers that can be used alone or inconjunction with the other disclosed treatment methods.

In a preferred aspect, the fluoropolymer layer is C-treated FEP that hasalso been primed and coated with LSR silicone by extrusion.

The layers may also be prelaminated together similar to that describedfor LIGHTSWITCH® Complete product (Saint-Gobain Performance PlasticsCorporation, Valley Forge, Pa.).

Total Organic Carbon (TOC) is the amount of carbon bound in an organiccompound and is often used as a non-specific indicator of pharmaceuticalmanufacturing equipment, among other things. TOC is utilized as aprocess control attribute in the biotechnology industry to monitor theperformance of unit operations that employ purification and distributionsystems.

In specific embodiments, TOC may be measured according to USPharmacopeia (USP) 643 and with equipment that utilizes a hightemperature wet oxidation reaction of UV-promoted chemical oxidation(Ultra-Clean Technology Handbook: Volume 1: Ultra-Pure Water, Ohmi,Tadahiro; CRC Press, 1993, pp. 497-517). Purified water is placed incontact with the polymer for 24 hours at 70° C., for example at a ratioof 3 cm² of article surface area to 1 mL of water. The water is removedfrom contact with the polymer and tested in a TOC analyzer. A suitablepiece of equipment is a TEKMAR DOHRMANN Model Phoenix 8000 TOC analyzer.

In particular embodiments, TOC may be measured for a container employedin a system of the disclosure including, for example by extraction froman internal surface area of the container (with results reflected asmg/cm² are for the TOC per square centimeter of the internal area). Inspecific embodiments, and merely as an example, the container may beextracted in purified water 70±2° C. for 24±2 hours. The extract may beanalyzed for TOC by converting TOC to carbon dioxide by acidificationand chemical wet oxidation with sodium persulfate, for example. Thecarbon dioxide liberated from the container may be measured using aninfrared detector. An example of an extraction ratio for a FEP containeris 3 cm²/mL (a typical extraction ratio). For some containers (such asFEP bags), no dilution is required because the level of TOC is less thanthe upper limit of a calibration curve, whereas for other embodiments(such as silicone tubing), dilution is required because of the levels ofthe TOC detected in the extract.

An example of TOC for a FEP container is 0.145 mg/L (0.00005 mg/cm² or0.001 mg/g). For embodiments that employ silicone tubing, extractionratios may be 14.6 cm²/mL (such as for Biosil) or may be 15.9 cm²/mL(such as for SR139), and an example of TOC for silicone Biosil tube is302 mg/L (0.021 mg/cm² or 0.023 mg/cm), and an example of TOC forsilicone SR139 tubing is 120 mg/L (0.008 mg/cm² or 0.0009 mg/cm). In atleast certain silicone tubing embodiments, the samples may be diluted,as the volume and concentration of the extraction cause the value to beabove the maximum detection of the machine. The dilution and differentextraction ratio requires the comparison of these samples with the bagsamples to be made on the weight/area value basis instead

One of skill in the art recognizes that TOC values may be characterizedin weight/volume. However, persons of skill in the art acknowledge thatratios for the container (particularly a FEP bag material) vs. ratiosfor silicone tubing are distinguishable; silicone tubing values can onlybe considered on a mg/cm² starting basis, as this value is independentof extraction ratio/dilution. One of skill in the art can calculate a“normalized” weight/volume ratio using a weight/area result as a basisand assuming a standard 3 cm²/mL extraction ratio (as an example) inorder to compare values on a weight/volume value.

In specific embodiments, the TOC of thermoplastic elastomers (TPE) is0.002 mg/cm² (0.032 mg/g or 5.88 mg/mL). In certain embodiments, the TOCof FEP is 0.00005 mg/cm² of interior wetted surface of an article (0.001mg/g or 0.145 mg/mL of article). In specific embodiments, the TOC ofsilicone is 0.021 mg/cm² or 63 mg/mL on interior wetted surface of anarticle. In a specific embodiment, a TOC for a PMP film is 0.07 ppm(0.00001 mg/cm²).

In specific embodiments, the TOC of fluorinated ethylene propylene (FEP)is 0.00005 mg/cm² (0.001 mg/g); the TOC of silicone materials, such assilicone tubing, is 0.021 mg/cm² (0.023 mg/cm) and 0.008 mg/cm² (0.009mg/cm); the TOC for a historically used cell culture bag is 0.002 mg/cm²(0.032 mg/g).

One of skill in the art recognizes that TOC values may be comparedacross different extraction ratios/dilutions if mg/cm² units areemployed. If units are mg/L, an extraction ratio must be known. Aconversion may occur as follows: the machine outputs a value in mg/L,dilution is factored in, and then this number is converted to mg/cm²using the surface area and total volume to extract. An example forSilicone Biosil is provided: Silicone Biosil Sample: 302 mg/L*1 L/1000mL*23.7 mL/347 cm²=0.021 mg/cm²

In a particular embodiments, TOC is compared in mg/cm² units because theextraction ratio or any dilution is not needed.

Below is an example of TOC calculation on a Silicone Tube Biosil sampleand on Silicone Tube SR139 sample.

Test Article Extraction Volume of Internal Surface Purified Sample Area(cm²) Length (cm) Water (mL) Silicone Tube 347 314 23.7 Biosil SampleSilicone Tube 342 295 21.5 SR139 Sample

Results for TOC Analysis Detection Sample mg/L mg/cm² mg/cm Limit (mg/L)Silicone Tube 302 0.021 0.023 0.1 Biosil Sample Silicone Tube 120 0.0080.009 0.1 SR139 Sample

Below is an example of TOC calculation on a FEP bag and on a singlelayer bag mostly composed of a SEBS (styrene block copolymer) but alsomay contain EVA and PP):

Test Article Extraction Internal Volume of Surface Purified Sample Area(cm²) Weight (g) Water (mL) FEP Bag 650 30.9 217 SEBS Bag 362.9 22.5 121

Results for TOC Analysis Detection Sample mg/L mg/cm² mg/g Limit (mg/L)FEP Bag 0.145 0.00005 0.001 0.1 SEBS Bag 5.88 0.002 0.032 0.1

In specific embodiments, a container comprises an inner surfacecomprising a polymer having a total organic carbon (TOC) in water ofless than 1 mg/cm², 0.9 mg/cm2, 0.8 mg/cm², 0.7 mg/cm², 0.6 mg/cm², 0.5mg/cm², 0.4 mg/cm², 0.3 mg/cm², 0.2 mg/cm², 0.1 mg/cm², 0.09 mg/cm²,0.08 mg/cm², 0.07 mg/cm², 0.06 mg/cm², 0.05 mg/cm², 0.04 mg/cm², 0.03mg/cm², 0.02 mg/cm², 0.01 mg/cm², 0.009 mg/cm², 0.008 mg/cm², 0.007mg/cm², 0.006 mg/cm², 0.005 mg/cm², 0.004 mg/cm², 0.003 mg/cm², 0.002mg/cm², 0.001 mg/cm², and so forth, or is nondetectable.

In particular embodiments, the TOC in water is less than an amount in arange from 0.001 mg/cm² to 0.1 mg/cm², 0.001 mg/cm² to 0.095 mg/cm²,0.001 mg/cm² to 0.075 mg/cm², 0.001 mg/cm² to 0.05 mg/cm2, 0.001 mg/cm²to 0.01 mg/cm2, 0.001 mg/cm² to 0.005 mg/cm², or 0.001 mg/cm² to 0.025mg/cm². In particular embodiments, the TOC in water is less than anamount in a range from 0.01 mg/cm² to 0.1 mg/cm², 0.01 mg/cm² to 0.075mg/cm², 0.01 mg/cm² to 0.05 mg/cm², or 0.01 mg/cm² to 0.025 mg/cm². Inparticular embodiments, the TOC in water is less than an amount in arange from 0.05 mg/cm² to 0.1 mg/cm², 0.05 mg/cm² to 0.09 mg/cm², 0.05mg/cm² to 0.075 mg/cm², or 0.05 mg/cm² to 0.06 mg/cm² In particularembodiments, the TOC in water is less than an amount in a range from0.005 mg/cm² to 0.1 mg/cm², 0.005 mg/cm² to 0.095 mg/cm², 0.005 mg/cm²to 0.075 mg/cm², 0.005 mg/cm² to 0.05 mg/cm², 0.005 mg/cm² to 0.025mg/cm², or 0.005 mg/cm² to 0.01 mg/cm².

The materials used in the oxygen permeable films of the currentembodiments may have a defined permeability. The permeability of theoxygen permeable elastomer films is at least 100 cc/m² per day,preferably at least 500 cc/m² per day, preferably at least 1000 cc/m²per day, preferably at least 1500 cc/m² per day, and most preferably atleast 2000 cc/m² per day and even more preferably at least 2200 cc/m²per day. The permeability of the oxygen permeable elastomerfluoropolymer composite films is at least 100 cc/m² per day, preferablyat least 500 cc/m² per day, preferably at least 1000 cc/m² per day,preferably at least 1500 cc/m² per day, and most preferably at least2000 cc/m² per day and even more preferably at least 2200 cc/m² per day.Oxygen permeability is measured with a MOCON Ox-tran 2/21H OxygenAnalyzer, following ASTM D3985, at 25° C. In another aspect of filmpermeablility, normalized units (cc-mm/m²-day) can be used to show afilm of any thickness. For example, the converted range for a 5 mil filmwould be from about 12.7 cc-mm/m²-day to at least about 279 cc-mm/m²-dayat a temperature of 25° C. The permeability of the contruct/compositecan stay in cc/m² terms as it would be comprised of two layers.

FIG. 2. is a view of a fluoropolymer coated elastomer cell culturecontainer 25. Fluoropolymer layer 10 (not shown) comprises the innerlayer of film 1 that is in contact with the cell culture. Fluoropolymercoated elastomer cell culture container 25 comprises at least onefluorinated elastomer film 1 bonded to form a cell culture compartment30 which is also bonded to at least one sealable access port 40 thatallows access to the interior of the container. Fluoropolymer coatedelastomer cell culture container 25 comprises a cell culture compartment30 which may be a bag, flask, or tube that comprises a closed systemthat also contains a least one sealable access port 40.

Bonding of film 1 to form cell culture container 30 and bonding ofaccess port 40 to the formed cell culture container 30 eachindividually, are accomplished by RF welding, ultrasonic welding, hotbar welding, chemical bonding, adhesive bonding, thermal fusion bonding,solvent welding, laser welding, corona discharge, radiation, laminationsuch as by extreme heat or melt lamination, etching, plasma treatment,wetting, adhesives, radiation, extrusion, co-extrusion, or combinationsthereof. Preferred methods of surface treatment include C-treatment(Saint-Gobain Performance Plastics Corporation, U.S. Pat. No.6,726,979), corona discharge, plasma treatment, etching, or combinationsthereof. Surface treatments may also involve chemical treatment withadditives or primers that can be used alone or in conjunction with theother disclosed treatment methods. Bonding of access port 40 may occurbefore, after, or during formation of cell culture container 30.

Cell culture container 30 comprises at least one access port 40 thatallows access to the interior of the container. Access port 40 maycontain a sealable lid or screw cap, a puncturable material or septa, ora valve assembly to permit access by a tube or syringe. A disposablesensor to measure pH/DO can also be included in the container to monitorcell culture conditions.

The location of the access port(s) is not restricted to the currentconfiguration and a port may be positioned for convenience of use ormanufacture. The access port(s) comprise fluoropolymer tubes that areattached to the composite container by RF welding, ultrasonic welding,hot bar welding, chemical bonding, adhesive bonding, thermal fusionbonding, solvent welding, laser welding, corona discharge, radiation,lamination such as by extreme heat or melt lamination, etching, plasmatreatment, wetting, adhesives, radiation, co-extrusion, or combinationsthereof.

Cell culture container 30 volumes may range from 1 mL to 50 L, 50 mL to10 L, but preferably 50 mL to 1 L.

Suitable growth media include, for example a nutrient or Lysogeny brothwith added hormones and/or growth factors.

Suitable cells include, for example connective tissue cells, skeletalcells, cardiac cells, epithelial cells, neural cells, endocrine cells,immune cells, lymphocytes, melanocytes, tumor cells, or mixturesthereof.

In one embodiment, in a first paragraph (1), the present inventionprovides an oxygen permeable film comprising a first elastomer layer;and a second fluid contact layer having a total organic carbon (TOC) inwater of less than 1 mg/cm².

2. The film according to paragraph 1, wherein the second fluid contactlayer is a fluoropolymer layer, and wherein the first elastomer layerand second fluoropolymer layer are adhered together.

3. The film according to paragraph 1 or 2, wherein the first elastomerlayer comprises natural polyisopene rubber (NR), synthetic polyisoprenerubber (IR), polybutadiene rubber (BR), chloropene rubber (CR), butylrubber (IIR), halogenated butyl rubbers (CIIR, BIIR), styrene-butadienerubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber(HNBR), ethylene propylene rubber (EPM), ethylene propylene diene rubber(EPDM), epichlorohydrin rubber (ECO), polyacrylic rubber (ACM, ABR),silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ),fluoroelastomers (FKM, FEPM), perfluoroelastomers (FFKM), polyetherblock amides (PEBA), chlorosulfonated polyethylene (CSM), ethylene-vinylacetate (EVA), cyclic olefin copolymers, polyolefin elastomers,elastomeric PET, or mixture thereof.

4. The film according to paragraph 1 or 2, wherein the first elastomerlayer is polymethylpentene polymer (PMP).

5. The film according to paragraph 1 or 2, wherein the first elastomerlayer is silicone rubber.

6. The film according to paragraph 5, wherein the silicone rubber ishigh consistency rubber (HCR), fluorosilicone rubber (FSR), liquidsilicone rubber (LSR), room temperature vulcanized rubber (RTV),thermoplastic silicone rubber (TPE), platinum cured silicone rubber, orperoxide cured silicone rubber.

7. The film according to paragraph 6, wherein the silicone rubber isthermoset, cured, vulcanized, catalyzed, or injection molded.

8. The film according to any one of paragraphs 2 to 7, wherein thesecond fluoropolymer layer is fluorinated ethylene-propylene (FEP),polytetrafluoroethylene (PTFE), polyvinylidenefluoride (PVDF),tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidenedifluoride (PVF), polychlorotrifluoroethlylene (PCTFE),tetrafluoroethylene/hexafluoropropylene/ethylene copolymer (HTE),chlorotrifluoroethylene/vinylidenefluoride copolymer,chlorotrifluoroethylene/hexafluoropropylene,ethylene/chlorotrifluoroethylene copolymers (ECTFE),ethylene/trifluoroethylene copolymers, ethylene/tetrafluoroethylenecopolymers (ETFE), tetrafluoroethylene/propylene copolymers (TFE/P),tetrafluoroethylene/hexafluoropropylene copolymers (FEP/HFP),hexafluoropropylene/tetrafluoroethylene/vinylidene copolymer (THV), orperfluoro(1-butenyl vinyl ether) homocyclopolymer having functionalizedpolymer-end groups

9. The film according to any one of paragraphs 2 to 8, wherein the firstelastomer layer and the second fluoropolymer layer are adhered togetherby bonding.

10. The film according to paragraph 9, wherein the bonding is bychemical bonding, adhesive bonding, thermal fusion bonding, solventbonding, laser welding, surface treatment, extrusion, co-extrusion,coating, lamination, or combinations thereof.

11. An oxygen permeable container comprising an outer elastomer layer;and an inner fluid contact layer having a total organic carbon (TOC) inwater of less than 1 mg/cm².

12. The container according to paragraph 11, wherein the fluid contactlayer is a fluoropolymer layer, and wherein the inner fluoropolymerlayer is adhered to the outer elastomer layer of the container.

13. The container according to paragraph 11 or 12, wherein the outerelastomer layer comprises natural polyisopene rubber (NR), syntheticpolyisoprene rubber (IR), polybutadiene rubber (BR), chloropene rubber(CR), butyl rubber (IIR), halogenated butyl rubbers (CIIR, BIIR),styrene-butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenatednitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylenepropylene diene rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylicrubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber(FSR, FVMQ), fluoroelastomers (FKM, FEPM), perfluoroelastomers (FFKM),polyether block amides (PEBA), chlorosulfonated polyethylene (CSM),ethylene-vinyl acetate (EVA), cyclic olefin copolymers, polyolefinelastomers, elastomeric PET, or mixtures thereof.

14. The container according to paragraphs 11 or 12, wherein the outerelastomer layer is polymethylpentene polymer (PMP).

15. The container according to paragraph 11 or 12, wherein the outerelastomer layer is silicone rubber.

16. The container according to paragraph 15, wherein the silicone rubberis high consistency rubber (HCR), fluorosilicone rubber (FSR), liquidsilicone rubber (LSR), or room temperature vulcanized rubber (RTV),thermoplastic silicone rubber (TPE), platinum cured silicone rubber, orperoxide cured silicone rubber.

17. The container according to paragraph 16, wherein the silicone rubberis thermoset, cured, vulcanized, catalyzed, or injection molded.

18. The container according to any one of paragraphs 12 to 17, whereinthe inner fluoropolymer layer is fluorinated ethylene-propylene (FEP),polytetrafluoroethylene (PTFE), polyvinylidenefluoride (PVDF),tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidenedifluoride (PVF), polychlorotrifluoroethlylene (PCTFE),tetrafluoroethylene/hexafluoropropylene/ethylene copolymer (HTE),chlorotrifluoroethylene/vinylidenefluoride copolymer,chlorotrifluoroethylene/hexafluoropropylene,ethylene/chlorotrifluoroethylene copolymers (ECTFE),ethylene/trifluoroethylene copolymers, ethylene/tetrafluoroethylenecopolymers (ETFE), tetrafluoroethylene/propylene copolymers (TFE/P),tetrafluoroethylene/hexafluoropropylene copolymers (FEP/HFP),hexafluoropropylene/tetrafluoroethylene/vinylidene copolymer (THV), orperfluoro(1-butenyl vinyl ether) homocyclopolymer having functionalizedpolymer-end groups

19. The container according to any one of paragraphs 12 to 18, whereinthe outer elastomer layer and the inner fluoropolymer layer are adheredtogether by bonding.

20. The container according to paragraph 19, wherein the bonding is bychemical bonding, adhesive bonding, thermal fusion bonding, solventbonding, laser welding, and surface treatment, extrusion, co-extrusion,coating, lamination, or combinations thereof.

21. The container according to any one of paragraphs 11 to 20, whereinthe container comprises a bag, flask, or tube.

22. The container according to paragraph 21, wherein the bag, flask, ortube contains at least one sealable access port.

23. A method to cultivate cells in an oxygen permeable containercomprising the steps of: a) adding media to the container; and b) addingcells, optionally with media, to the container, wherein the containercomprises an outer elastomer layer, and an inner fluid contact layerhaving a total organic carbon (TOC) in water of less than 1 mg/cm².

24. The method according to paragraph 23, where the inner fluid contactlayer is a fluoropolymer layer, and wherein the inner fluoropolymerlayer is adhered to the outer elastomer layer of the container.

25. The method according to paragraph 23 or 24, wherein the outerelastomer layer is natural polyisopene rubber (NR), syntheticpolyisoprene rubber (IR), polybutadiene rubber (BR), chloropene rubber(CR), butyl rubber (IIR), halogenated butyl rubbers (CIIR, BIIR),styrene-butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenatednitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylenepropylene diene rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylicrubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber(FSR, FVMQ), fluoroelastomers (FKM, FEPM), perfluoroelastomers (FFKM),polyether block amides (PEBA), chlorosulfonated polyethylene (CSM),cyclic olefin copolymers, polyolefin elastomers, elastomeric PET, andethylene-vinyl acetate (EVA).

26. The method according to paragraph 23 or 24, wherein the outerelastomer layer is polymethylpentene polymer (PMP).

27. The method according to paragraph 23 or 24, wherein the outerelastomer layer is silicone rubber.

28. The method according to paragraph 27, wherein the silicone rubber ishigh consistency rubber (HCR), fluorosilicone rubber (FSR), liquidsilicone rubber (LSR), or room temperature vulcanized rubber (RTV),thermoplastic silicone rubber (TPE), platinum cured silicone rubber, orperoxide cured silicone rubber.

29. The method according to paragraph 28, wherein the silicone rubber isthermoset, cured, vulcanized, catalyzed, or injection molded.

30. The method according to one of any paragraphs 24 to 29, wherein theinner fluoropolymer layer is fluorinated ethylene-propylene (FEP),polytetrafluoroethylene (PTFE), 3M™ Dyneon™ TFM™ modified PTFE,polyvinylidenefluoride (PVDF), tetrafluoroethylene-perfluoro(propylvinyl ether) (PFA), polyvinylidene difluoride (PVF),polychlorotrifluoroethlylene (PCTFE),tetrafluoroethylene/hexafluoropropylene/ethylene copolymer (HTE),chlorotrifluoroethylene/vinylidenefluoride copolymer,chlorotrifluoroethylene/hexafluoropropylene,ethylene/chlorotrifluoroethylene copolymers (ECTFE),ethylene/trifluoroethylene copolymers, ethylene/tetrafluoroethylenecopolymers (ETFE), tetrafluoroethylene/propylene copolymers (TFE/P),tetrafluoroethylene/hexafluoropropylene copolymers (FEP/HFP),hexafluoropropylene/tetrafluoroethylene/vinylidene copolymer (THV), orperfluoro(1-butenyl vinyl ether) homocyclopolymer having functionalizedpolymer-end groups.

31. The method according to one of any paragraphs 24 to 30, wherein theouter elastomer layer and the innner fluoropolymer layer are adheredtogether by bonding.

32. The method according to paragraph 31, wherein the bonding is bychemical bonding, adhesive bonding, thermal fusion bonding, solventbonding, laser welding, and surface treatment, extrusion, co-extrusion,coating, lamination, or combinations thereof.

33. The method according to one of any paragraphs 23 to 32, wherein thecontainer comprises a bag, flask, or tube.

34. The method according to paragraph 33, wherein the bag, flask, ortube contains at least one sealable access port.

35. The method according to paragraph 23, wherein steps a) and b) areinterchangeable.

36. The method according to paragraph 23, further comprising the step ofincubating the container.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Tested Samples

TABLE 1 TOC data Extraction TOC Material Form Conditions (TOC-Blank) PMPFilm Film 70° C., 24 0.07 ppm hours, 3 (0.00001 cm²/mL mg/cm²) PrimedBag with 70° C., 24 0.02 ppm Thermoset Laminate as hours, 3 (0.00001Silicone/1 mil bottom sheet cm²/mL mg/cm²) FEP Laminate Unprimed Bagwith 70° C., 24 0.01 ppm Thermoset Laminate as hours, 3 (0.000003Silicone/1 mil bottom sheet cm²/mL mg/cm²) FEP Laminate PMP/1 mil Bagwith 70° C., 24 0.01 ppm FEP Laminate Laminate in hours, 3 (0.000003cut-out bag cm²/mL mg/cm²) form (i.e. portion of bottom sheet)Thermoplastic Bag with 70° C., 24 0.36 ppm Silicone/1 mil Laminate inhours, 3 (0.0001 FEP Laminate cut-out bag cm²/mL mg/cm²) (with liner)form (i.e. portion of bottom sheet) Thermoplastic Bag with 70° C., 243.59 ppm Silicone/1 mil Laminate in hours, 3 (0.001 FEP Laminate cut-outbag cm²/mL mg/cm²) (without liner) form (i.e. portion of bottom sheet)FEP 2PF-0290 FEP 70° C., 24 0.30 ppm “Incumbent” Bag hours, 3 (0.0001cm²/mL mg/cm²) ppm = mg/L

Example 2 Dissolved Oxygen Experiments

An incumbent FEP bag made from 5 mil FEP on top and bottom a and aprototype bag made from 5 mil FEP on top and thermoplastic silicone/FEPon the bottom were filled to 300 mL and 190 mL respectively with tapwater. These volumes correlate to a 1 cm thickness of water in the bagwhen it is lying flat, a recommendation that is well-known in industry.A port was cut out of each bag in order to feed in a nitrogen line topurge the water in the bag to a level below saturation. Dissolved oxygen(DO) levels were monitored using the PreSens probe affixed to theoutside of the bag and aligned with the sensor dot on the inside of thebag. When the DO levels in the water reached a reasonably low value(˜6-8%), the nitrogen line was removed from the bag and the port openingwas clamped shut. The bag and rack were returned to a horizontal restingposition for the remainder of the experiment. The PreSens probe wasprogrammed to take DO measurements at designated time points in order tomeasure the ingress of oxygen as a function of time. It was assumed thatthe only path for oxygen ingress was through the walls of the bags.

Results:

Measurements from the PreSens system were downloaded and plotted inExcel for both bags (FIGS. 3, 4, and 5). The data was studied andobservations were made to bracket the data. For instance, for theprototype bag, the beginning measurements were excluded because the bagwas moved and manipulated in order to remove an air bubble from abovethe sensor. Also, because a longer measurement was taken for theprototype bag, the data was split into two linear sections, “InitialIngress” and “Approach to Equilibrium”. Due to this, the best comparisonto make with the current data available is between FIG. 3 and FIG. 4which demonstrate the initial ingress of oxygen into each of the bags asa function of time when starting at similar DO levels (˜6-8%) for asimilar amount of time (˜68-70 min). Looking at FIG. 3 and FIG. 4, thecomparison of slopes can be made to understand the difference in rate ofingress between the incumbent and prototype bags. The incumbent bag as arate of 0.008% DO/min while the prototype bag has a rate of 0.06%DO/min, a difference of over 7×. This alludes to the positive impact ofthe more permeable material on the ingress of oxygen.

Although the present invention has been described with reference topreferred embodiments, persons skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention. All references cited throughout thespecification, including those in the background, are incorporatedherein in their entirety. Those skilled in the art will recognize, or beable to ascertain, using no more than routine experimentation, manyequivalents to specific embodiments of the invention describedspecifically herein. Such equivalents are intended to be encompassed inthe scope of the following claims.

What is claimed is:
 1. An oxygen-permeable film comprising: a firstpolymer layer having a thickness in the range of about 0.01 mm to about5 mm, the first polymer layer being a silicone layer; and a second fluidcontact layer having a thickness in the range of about 0.01 mm to about0.1 mm, the second fluid layer being an FEP layer, wherein the firstpolymer layer and the second fluid contact layer are adhered together,and wherein the oxygen permeable film has an oxygen permeability of atleast 2000 cc/m² per day.
 2. The oxygen-permeable film according toclaim 1, wherein the first polymer layer has a thickness in the range of0.1 to 1 mm.
 3. The oxygen-permeable film according to claim 1, whereinthe first polymer layer is a layer of cured liquid silicone rubber. 4.The oxygen-permeable film according to claim 1, wherein the firstpolymer layer is a layer of high consistency rubber.
 5. Theoxygen-permeable film according to claim 1, wherein the FEP layer has athickness in the range of 0.01 to 0.04 mm.
 6. The oxygen-permeable filmaccording to claim 1, wherein a primer is used in the adhesion of firstpolymer layer to the FEP layer.
 7. The oxygen-permeable film accordingto claim 1, wherein the FEP layer is C-treated at a surface adhered tothe first polymer layer.
 8. The oxygen permeable film according to claim1, wherein the FEP layer is C-treated at a surface adhered to the firstpolymer layer, wherein the first polymer layer is a layer of curedliquid silicone rubber, and wherein a primer is used in the adhesion offirst polymer layer to the FEP layer.
 9. The oxygen-permeable filmaccording to claim 1, wherein the FEP layer is treated at a surfaceadhered to the first polymer layer with a treatment selected from coronadischarge, plasma treatment, etching, and combinations thereof.
 10. Theoxygen-permeable film according to claim 1, wherein FEP layer has a TOCin water of less than 0.1 mg/cm².
 11. An oxygen-permeable containercomprising an oxygen permeable film that comprises: an outer polymerlayer having a thickness in the range of about 0.01 mm to about 5 mm,the outer polymer layer being a silicone layer; and an inner fluidcontact layer having a thickness in the range of about 0.01 mm to about0.1 mm, the inner fluid layer being an FEP layer, wherein the outerpolymer layer and the inner fluid contact layer are adhered together,and wherein the oxygen permeable film has an oxygen permeability of atleast 2000 cc/m² per day.
 12. The container according to claim 11,wherein the container is in the form of a bag, flask, or tube.
 13. Thecontainer according to claim 11, wherein the container is in the form ofa cell culture bag comprising the oxygen-permeable film as a wallthereof.
 14. The container according to claim 11, wherein the outerpolymer layer has a thickness in the range of 0.1 to 1 mm.
 15. Thecontainer according to claim 11, wherein the outer polymer layer is alayer of cured liquid silicone rubber.
 16. The container according toclaim 11, wherein the outer polymer layer is a layer of cured highconsistency rubber.
 17. The container according to claim 11, wherein theFEP layer has a thickness in the range of 0.01 to 0.04 mm.
 18. Thecontainer according to claim 11, wherein a primer is used in theadhesion of outer polymer layer to the FEP layer.
 19. The containeraccording to claim 11, wherein the FEP layer is C-treated at a surfaceadhered to the outer polymer layer.
 20. The container according to claim11, wherein the FEP layer is C-treated at a surface adhered to the outerpolymer layer, wherein the outer polymer layer is a layer of curedliquid silicone rubber, and wherein a primer is used in the adhesion ofouter polymer layer to the FEP layer.
 21. The container according toclaim 11, wherein the FEP layer is treated at a surface adhered to thefirst polymer layer with a treatment selected from corona discharge,plasma treatment, etching, and combinations thereof.
 22. The containeraccording to claim 11, wherein the container comprises at least onesealable access port.
 23. The container according to claim 9, whereinthe inner fluid contact layer has a TOC in water of less than 0.1mg/cm².
 24. A method to cultivate cells in an oxygen permeable containeraccording to claim 11, the method comprising: a) adding media and cellsto the container; and b) incubating the container.